Plastoquinone synthesis inhibition by tetrabromo biphenyldiol as a widespread algicidal mechanism of marine bacteria

Algae and bacteria have complex and intimate interactions in the ocean. Besides mutualism, bacteria have evolved a variety of molecular-based anti-algal strategies. However, limited by the unknown mechanism of synthesis and action of these molecules, these strategies and their global prevalence remain unknown. Here we identify a novel strategy through which a marine representative of the Gammaproteobacteria produced 3,3’,5,5’-tetrabromo-2,2’-biphenyldiol (4-BP), that kills or inhibits diverse phytoplankton by inhibiting plastoquinone synthesis and its effect cascades to many other key metabolic processes of the algae. Through comparative genomic analysis between the 4-BP-producing bacterium and its algicidally inactive mutant, combined with gene function verification, we identified the gene cluster responsible for 4-BP synthesis, which contains genes encoding chorismate lyase, flavin-dependent halogenase and cytochrome P450. We demonstrated that in near in situ simulated algal blooming seawater, even low concentrations of 4-BP can cause changes in overall phytoplankton community structure with a decline in dinoflagellates and diatoms. Further analyses of the gene sequences from the Tara Oceans expeditions and 2750 whole genome sequences confirmed the ubiquitous presence of 4-BP synthetic genes in diverse bacterial members in the global ocean, suggesting that it is a bacterial tool potentially widely used in global oceans to mediate bacteria-algae antagonistic relationships.


Figures S1 to S11
Legends for Tables S1 to S6 Other supporting materials for this manuscript include the following: Tables S1 to S6 Supplementary Materials

Isolation and identification of algicidal strains
Synechococcus sp.PCC7002 (obtained from the Pasteur Culture Collection) was cultured in A + medium at 26°C with a 12:12 h light:dark cycle of 50 μmol photons m −2 s −1 , used to isolate algicidal bacteria in this study.Sediment samples, often considered an important source of algicidal bacteria, were collected from the coast of Qingdao (N 36°09′, E120°21′).The bacterial suspensions were obtained by soaking and shaking the sediments in sterile seawater for 30 min and adding them to the exponentially growing Synechococcus sp.PCC7002 for one-week cocultivation.After observing algal lysis, the representative bacterial strains were isolated by serial dilution.The algicidal activity was verified by inoculating the bacteria on a Synechococcus plate and observing whether there was an algae-lysing zone.
The morphological characteristics of the algicidal strains were observed with an Olympus BX-51 optical microscope and a Hitachi H-7650 transmission electron microscope.Their classifications were confirmed by amplifying the 16S rRNA gene with primers 27F and 1492R.Here, an algicidal bacterial strain Microbulbifer sp.RZ01 (belonging to the class γ-proteobacterium) was isolated.In addition, another bacterial strain, TB12003, with the same 16S rRNA gene as RZ01 but with a different colony color, was isolated.

Determination of the growth curve of algicidal bacterial strain Microbulbifer sp. RZ01
Colonies grown on solid 2216E, were transferred to 5 mL liquid 2216E and activated at 28°C and 150 rpm for 24 h.Subsequently, the culture was inoculated into 150 mL liquid 2216E at a concentration of 1.0% (v/v), and incubated at 28°C and 150 rpm.The samples were collected every 4 h, and the OD at 600 nm was measured with a spectrophotometer.

Determining how strain RZ01 inhibits the algae and the properties of the algicidal substance
Strain RZ01 was grown to exponential, stationary and decline phases in 20-h, 40-h and 60-h incubations, respectively, after which it was inoculated at a concentration of 5% (v/v) into exponentially growing Synechococcus sp.PCC7002.Moreover, in order to To determine the anti-algal mode of the bacteria (i.e., whether the bacteria killed the algae directly or indirectly by secreting substances), the cell-free supernatant and cells of the RZ01 culture were added to the algal cultures to assess their algicidal activity.In brief, strain RZ01 was inoculated into 30 ml of 2216E broth and grown on a shaker at 150 rpm 28°C for 60 h.The cell-free supernatant was collected by centrifugation at 8,000 rpm for 10 min and then passed twice through a 0.22-μm filter (Millipore).The cell pellets were washed twice with sterile seawater and re-suspended in A + medium.These different fractions were then added into exponentially growing cultures of Synechococcus sp.PCC7002.The algicidal rate was determined as follows: Algicidal rate = (FC-FT)/FC x 100%, where FT and FC represent the fluorescence intensity of the algal culture with and without bacteria, respectively.All experiments were performed in triplicate.
To test the thermal stability of the algicidal substance, the cell-free supernatant was incubated at 100°C for 30 min, and then cooled to 28°C before it was added to Synechococcus cultures at a concentration of 10% (v/v).To determine the effect of pH on the algicidal substance, the cell-free supernatant was adjusted to pH 1, 3, 5, 7, 9, 11 or 13 using 1 M HCl and 1 M NaOH and incubated for 2 h.It was then adjusted back to the initial pH (pH 8), after which it was added to the Synechococcus culture at a concentration of 10% (v/v).Finally, to characterize the molecular size of the algicidal substance, the cell-free supernatant was sequentially passed through 30 KDa, 10 KDa and 3 KDa pore-size ultrafiltration tubes (Millipore) and centrifuged at 4,000 rpm for 30 min, after which the corresponding filtrates were added to the algal cultures.

Heterologous expression and enzymatic activity assay of homogentisate solanesyltransferase
The P. tricornutum homogentisate solanesyltransferase (HST) ORF (lacking a transit peptide sequence) was cloned into the pGEX-6P-1 vector for the expression of HST as a recombinant protein with an N-terminal glutathione S-transferase (GST)-tag, according to the manufacturer's instructions.Protein expression was carried out in E. coli Rosetta (DE3); these cells were grown at 37°C in LB medium supplemented with 25 μg/mL chloramphenicol and 100 μg/mL ampicillin.Expression was induced by the addition of IPTG at final concentration of 0.6 mM and the cells were cultured for 16 h at 20°C.Subsequently, the cells were collected by centrifugation and lysed with buffer comprising 8 M urea, 50 mM Tris, 300 mM NaCl, and 0.1% Triton X-100, pH 8.0 followed by sonication.The supernatant collected after the centrifugation step contained crude proteins, which were purified with an Ni-NTA column, separated by SDS-PAGE and identified by Western blot.Meanwhile, the E. coli cell membranes, (expressing hst), were collected from the IPTG-induced E. coli cultures by sequential centrifugation at 500 g and ultracentrifugation at 150,000 g.The mobile phase was acetonitrile and 0.1% acetic acid at a ratio of 65:35 (v/v).The flow rate was maintained at 1.0 mL/min, and the effluent was monitored using a fluorescence detector with excitation and emission wavelengths of 290 nm and 330 nm, respectively.

Comparison of Microbulbifer sp. strains RZ01 and TB12003
The color of colonies of Microbulbifer sp.RZ01 grown on solid 2216E was dark yellow, while Microbulbifer sp.TB12003 colonies were light yellow (Fig. S1A).In contrast to algicidal bacterial strain RZ01, TB12003 had lost inhibitory activity against algae (Fig. S1B).The inability of the Microbulbifer sp.TB12003 to synthesize 4-BP was confirmed by HPLC analysis of the crude extracts of both strains (Fig. S1C).